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AUTOPHAGY IN TB

Eun-Kyeong Jo, Jae-Min Yuk, Dong-Min Shin

CHAPTER 2B.7

In countries where TB is highly stigmatized, women with the disease may not be eligible for marriage or

forced to leave their children.

‘I sleep on the edge of an abyss.’

The NightEduardo Galeano

Below, I’m AwakeCirenaica MoreiraDigital print

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Autophagy is a catabolic process by which cytoplasmic cargos are sequestrated and degraded by fusion with autophagosomes and lysosomes. Macroautophagy (hereafter referred to as autophagy), is an essential intracellular process that enables cells to auto-digest their own substances when metabolically stressed, and remove large protein aggregates that cannot be cleared by proteasomal degradation. In recent years, the roles of autophagy have expanded to include cell-defense effectors in innate immunity (1, 2). Numerous reports have described a key role for autophagy in host defense against diverse bacterial, viral, and parasitic infections (2). Thus, autophagy is now being recognized as a crucial cellular process that sanitizes the cytoplasm by removing intracellular microbes (3). Because of its ability to degrade foreign parasites and participate in antigen presentation, autophagy is thought to play a bridge role between innate and adaptive immunity.

MTB one of the most successful human pathogens, still causes active TB disease in millions of people worldwide. The ability of the tubercle bacillus to cause high morbidity and mortality depends on its persistence in the intracellular niche by avoiding phagosomal maturation (4). A principal survival tactic of pathogenic MTB is its subversion of the endocytic/phagocytic pathway inside host cells to avoid fusion with lysosomes (5). We have recently learned that autophagy activation is a key means of eradicating MTB through overcoming MTB phagosomal maturation (6). Efforts to understand the mechanisms by which autophagy contributes to the eradication of intracellular bacteria would open a new avenue in the treatment and in the prevention of TB (6). Moreover, vaccines that effectively induce autophagy would represent an innovative strategy for preventing both new mycobacterial infections and reactivation of latent TB (7). Thus, therapeutic and vaccine intervention in the autophagy pathway may be a promising approach for future pre-clinical and clinical trials.

While recent evidence indicates autophagy as a crucial anti-mycobacteria host defense mechanism, it is also apparent that autophagy pathways can crosstalk with a variety of innate immune signaling pathways in response to mycobacteria or their components. In the current article, we discuss the roles, mechanisms, and evidence

AUTOPHAGY IN TB CHAPTER 2B.7

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310 SECTION 2 MAIN PLAYERS

underlying these multi-faceted roles of autophagy in mycobacterial infection. In addition, the crosstalk between autophagy and innate pathways will be discussed. In the �nal part of this review, we will summarize new insights into the autophagic link between the innate and adaptive immune responses, which may result in vaccine-mediated and therapeutic manipulation of autophagy against TB. These efforts will make the targeting of autophagy for ameliorating human TB possible.

INTRODUCTIONDespite global efforts to combat TB, it remains a major public health problem with increasing multi- and high level-drug resistance worldwide. The causative pathogen of TB is MTB, a successful human pathogen that can survive in the infected host for a lifetime. MTB uses multiple strategies to resist and/or escape from host defensive mechanisms upon infection of mammalian cells. The conventional modalities for controlling TB have had little impact on the current TB epidemic. Potential solutions to this problem include the development of new and effective strategies for drug and vaccine development. From this perspective, the induction of autophagy, an emerging host defense pathway, has important role(s) in antimycobacterial immunity that may be crucial in future clinical applications. Autophagy activation has at least two functions in mycobacterial infection. Firstly, it is an important effector mechanism for the eradication of these intracellular pathogens. Secondly, the autophagy pathway is fundamentally important in maintaining intracellular homeostasis during infection, while avoiding host damage by excessive in�ammatory responses. Autophagy thus, acts to maintain the balance in interactions between the host and pathogen, thus preventing further exacerbations of human TB.

Earlier studies showed that autophagy stimulation up-regulates the phagosomal co-localization of Beclin-1, an essential molecule for autophagy activation, resulting in the suppression of intracellular survival of mycobacteria (8). Th1 cytokine interferon (IFN)-γ or transfection with LRG-47, an effector of IFN-γ, can induce autophagy, which promotes antimycobacterial action (8). In addition, human immunity-related p47 guanosine triphosphatases (IRG) were found to play a role in the induction of autophagy and caused a reduction in intracellular MTB load (9). Some members of the IFN-γ–inducible GTPase superfamily, such as guanylate-binding proteins (Gbps), are essential autophagy host defense proteins and confer immunity to listerial and mycobacterial infection (10).

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311AUTOPHAGY IN TB CHAPTER 2B.7

Vitamin D and vitamin D receptor (VDR) signaling activation have also been reported as an important modulator of human innate immune responses against mycobacterial infection (11). Recent studies have focused on the role of vitamin D in the activation of autophagy in human macrophages (12). Vitamin D-induced autophagy enhances antimycobacterial activity through human cathelicidin CAP-18/LL-37 (12). Local synthesis of 1,25D in innate cells in response to TLR signaling appears to induce autophagy through 25-hydroxyvitamin D3 1-a-hydroxylase (Cyp27b1) expression and functional vitamin D signaling (13). Moreover, interleukin (IL)-1 and innate immune activation have been recognized to be associated with autophagy pathways in mycobacterial infection. A new group of innate immunity receptors, including the sequestasome (p62/SQSTM1), serve as autophagy adaptors and recognize and capture intracellular microbes through a microtubule associated protein light chain 3 (LC3) interacting region (14).

This review will highlight our current understanding of autophagy in mycobacterial infection. Recently, our understanding of how innate and adaptive immune responses crosstalk with autophagy pathways has greatly broadened. Innate immune signaling pathways will be discussed here, in the context of regulation of autophagy pathways during mycobacterial infection. Additionally, recent progress has elucidated Th1/Th2 cytokine modulation, vitamin D-mediated autophagy activation, and antimicrobial mechanisms involving p62/sequestasome-like receptors through the generation of neoantimicrobial peptides that enhance antibacterial effects against mycobacteria in phagosomes. These efforts will contribute to the development of novel anti-TB therapeutics.

A BRIEF OVERVIEW OF AUTOPHAGY PATHWAY As brie�y mentioned above, autophagy is a highly-conserved eukaryotic intracellular pathway for the maintenance of homeostasis against a variety of intracellular stresses. Autophagy is initiated by the formation of crescent-shaped autophagic isolation membranes (phagophores) that expand to form a double-membrane vesicle, the autophagosome. The third step of the autophagy pathway is maturation, during which the outer autophagic membrane of autophagosomes fuses with the lysosomal membrane, resulting in the formation of autolysosomes that can eventually degrade their contents (Figure 2B.7.1).

More than 30 autophagy-related (ATG) genes have been identi�ed in yeast (15). The orthologs of the ATG gene products have also been reported to be involved in autophagy regulation in higher eukaryotes, including humans (15). The roles of

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each ATG gene have been described; these include functions in the regulation of initiation, vesicle nucleation, cargo packaging, vesicular expansion, and recycling during the autophagic process (16).

Amphisome

AutolysosomeAAutolysosome

1. Initiation

Phagophore

ER

Beclin 1Atg14L

VPS34

VPS15

Atg14L-Beclin 1complex

PI3P

Atg5/12/16 6 6 6666

LC3 II

2. Elongation, Completion

Autophagosome

PI3Pphosphatase

Lysosome

3. Maturation

Beclin 1UVRAG

VPS34

VPS15

UVRAG-Beclin 1complex

PI3P

PI3Pphosphatase

UVRAG

C-Vps

Rab7

Rab7

Rubicon

Autophagosommaturation

UVRAG complex

Rubicon complex

WIPI 1/2

LC3 mAtg4

Atg7 Atg10

Atg3Atg7

Figure 2B.7.1 A schematic overview of the autophagy pathway

Note: Autophagy can be divided through the following stages including initiation, elongation and completion of the phagophore, autophagosome maturation, and formation of autolysosome. (1) Initiation: Autophagic isolation membrane or phagophore is mainly generated from the ER membrane through phosphatidylinositol 3-phosphate (PI3P) produced by the phosphatidylino-sitol 3-kinase hVPS34 in a complex with Atg14L, Beclin 1, and VPS15. The mammalian Atg18 paralogs WIPI 1/2 is downstream molecules of PI3P, which bind PI3P, and facilitate the formation of the LC3-positive membrane. PI3P phosphatases that inhibit activation of PI3P and Atg4 paralogs

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that catalyze LC3 delipidation is negative regulator of autophagy initiation. (2) Elongation and completion: Autophagic isolation membrane is elongated to form the phagophore structures based on two conjugation systems. (a) Covalent Atg5 and Atg12 complex noncovalently conjugate with Atg 16 and function as an E3-like enzyme. (b) Atg5/12/16 complex enzumatically induces the conversion of LC3 to LC3 II. A series of steps play crucial role in the elongation and completion, to form the double membrane autophagosome. (3) Autophagosome fuses with late endosomal and lysosomal organelles in the maturation stage and makes intermediates called amphisome, eventually producing a fully lytic organelle termed autolysosome. The formation of Amphisome is dependent on beclin 1 (UVRAG-Beclin 1 complex) via PI3P activation or independent on beclin 1 (UVRAG-C Vps-Rab7 complex) via Rab7 GTPase activation. Rubicon-Rab7 complex inhibits autophagosome maturation. The single-membraned autolysosome contains the degradation products of captured targets.

The initiation of autophagy activation involves the formation of beclin 1 complex and its associated proteins, such as vacuolar sorting protein 34 (VPS34), a class III phosphatidylinositol-3 kinase (PI-3K), UV irradiation resistance-associated gene (UVRAG) and Atg14L (17). Recent studies have shown that Atg14 is required for the initiation of autophagic membrane biogenesis through the mediating localization of the autophagy-speci�c PI-3K complex to the endoplasmic reticulum, which leads to the appearance of phosphatidylinositol 3-phosphate (PI3P) in the ER and the initiation of double membrane vesicle formation (18). Current studies are focused on determining the physiologic and coordinating functions of the beclin 1 complex that initiates the autophagic process (17). Notably, beclin 1-VPS34 complexes play a pleiotropic role in the autophagy control and membrane traf�cking processes (16, 17).

In the elongation step, two ubiquitin-like conjugation cascades are proposed to play a key role in the extension of autophagic vesicle membranes (Figure 2B.7.1). The Atg12-Atg5 conjugate interacts non-covalently with Atg16L and forms a large multimeric complex called the Atg16L complex, which acts as an E3-like enzyme. The Atg5/12/16 complex drives the lipidation and conjugation of LC3/Atg8 to the autophagosomal membrane in a reaction that requires Atg3/7 complex (19). The lipidated form of LC3 (LC3-II) remains within the autophagosome lumen, but is removed from the autophagosome membrane after fusion with lysosomes; LC3-II is then degraded (20). Although much less is known of the molecular mechanisms of autophagosome closure through fusion with lysosomes, Atg8/LC3 is thought to play an important role in the closure of the forming autophagosome (19). The

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targeting of autophagosomes to lysosomes in the perinuclear region occurs in a microtubule- and dynein-dynactin motor complex-dependent manner (21).

Finally, the maturation of the autophagosome occurs through the fusion of the autophagic membrane with the late endosomal and lysosomal organelles. This results in the dissolution of the inner membrane of the autophagosome and the formation of the autolysosome (14, 22). The autophagosome/endosome maturation and degradation of endocytic captured material can be mediated through the interaction of the Beclin1-binding autophagic tumour suppressor, UVRAG, with the class C Vps complex (23). Other molecules, including the lysosomal membrane protein, lysosomal associated membrane protein (LAMP-2), the small GTPase Rab7 (mammalian homologue of yeast Ypt7), AAA ATPase SKD1, Hrs, and soluble N-ethylmalemide-sensitive factor attachment protein receptor (SNARE) protein Vti1, have been characterized and are involved in the autophagic maturation and endosomal transport in mammalian cells (22, 24, 25, 26, 27). A speci�c PI3P phosphatase Jumpy ⁄ MTMR14 has been found to modulate the maturation phase of autophagy (28). Accumulating evidence suggests that these molecules contribute to the multi-tiered connections between autophagosomal maturation and endocytic traf�cking, resulting in the enhancement of the delivery of endocytic/autophagic cargo into the degradative compartments (23). Further studies are needed to identify and characterize the roles of these molecules in the regulation of autophagic maturation.

‘XENOPHAGY’ AS AN EFFICIENT MYCOBACTERIA ERADICATION SYSTEM The autophagy pathway, an essential self-eating mechanism, is involved in the bulk digestion of cytoplasmic constituents through the fusion of the outer autophagosomal membrane with a catalytic chamber, the lysosome (Figure 2B.7.2). Through this major physiological function, the autophagy pathway can remove unwanted constituents, and recycle cytoplasmic material to maintain energy homeostasis during stressful conditions. Recently, accumulating evidence has provided the roles of autophagy, not only its contribution to the removal of cytoplasmic constituents, but also its ability to target foreign pathogenic invaders for degradation during infection (29). Therefore, the use of the autophagy pathway to digest foreign intracellular pathogens has emerged as a central part of the host defense system, referred as ‘xenophagy (antimicrobial autophagy)’ (30).

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The ability of mycobacteria to modulate the endocytic pathway ensures MTB persistence in recycling endocytic vesicles and confers resistance to microbicidal autophagic processes (31). The host small GTPases Rab5 and Rab7 are key components in the dynamics of cargo progression between early and late endocytic organelles (32). PI-3P is critically involved in the process of autophagy and is the product of host protein type III PI-3K hVPS34, which is a major Rab effector and essential for autophagy activation (33). Pathogenic MTB can block Rab conversion on phagosomes, thereby eliciting the arrest of MTB phagosome maturation (33). Additionally, MTB can elaborate mycobacterial factors, including lipoarabinomannan or enzyme SapM (a PI3P phosphatase) that act upon PI3P and interfere with phagosome maturation (33). The induction of autophagy results in the activation/mobilization of hVPS34, thereby enhancing PI3P production that can override the MTB block of phagolysosome biogenesis (34).

Multiple strategies by pathogenic mycobacteria have been reported to contribute to their persistence in macrophage phagosomes through the modulation of host defense machineries. Besides PI3K hVPS34, the Ser/Thr kinase target of rapamycin (Tor) is an important regulator of autophagy. Tor acts as a central regulator of cell growth and is itself regulated by a wide range of signals, including growth factors, nutrients and stress conditions, energy status, and other stresses (35). MTB activation of the host mTOR/S6K1 pathway may contribute to the inhibition of autophagy effector pathways (36). The pharmacologic inhibition of the mTOR pathway by rapamycin induces autophagy in macrophages and enhances intracellular mycobacterial killing activity (8).

Host professional phagocytes and innate cells have sophisticated strategies to combat invading mycobacteria (31). In several animal studies, ATG have been shown to play an essential role in host protection against mycobacteria, as well as other viral and intracellular bacterial pathogens (8, 12, 37). In human macrophages, vitamin D-induced antimicrobial activities are dependent on Beclin-1 and Atg5 expression (12). Moreover, the autophagy pathway can kill intracellular microbes directly through the delivery of bactericidal peptides to intracellular pathogens (38).

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Figure 2B.7.2 A schematic diagram of the xenophagy as a clearance system against mycobacteria

Note: Xenophagy is a novel strategy of antimicrobial effects and mediates anti-mycobacterial resistance. Several mechanisms are thought to contribute to the xenophagy-induced antimicrobial effect against mycobacteria. Firstly, the sensing of a number of innate receptors (also known as pattern recognition receptors, PRRs), which includes TLRs, RLRs, and NLRs, with their cognate ligands activates autophagy that plays a crucial role for the elimination of intracellular mycobacteria (called ‘xenophagy’). Several downstream signalling molecules of PRRs are shown to participate in the activation of autophagy. On the other hand, PRRs-induced NFκB activation inhibits autophagy in host cells. Secondly, the VDR signaling induces the activation of antimicrobial peptides and the activation of autophagy. TLR2/1-dependent signals and 1,25D3 mediates antimycobacterial activity through the induction of cathelicidin and DEFB4 expression. Vitamin D3-induced upregulation of cathelicidin is required for the autophagosome formation and phagolysosomal fusion in human macrophages. Thirdly, the production of TNF-α and IFN-γ (Th1 responses) through mycobacterial antigen-induced PRRs activation upregulates autophagy, whereas the secretion of IL-4 and IL-13 (Th2 responses) downregulates autophagy. Lastly, xenophagy is known to cross-talk between ubiquitination pathways, contributing to the selective autophagosomal degradation of ubiquitinated target proteins. Autophagy receptors p62/SQSTM1, NBR1 and NDP52 reveal that these cargo receptors or adaptors for the autophagic degradation of ubiquitinated substrates contribute to the selective autophagosomal degradation of ubiquitinated target proteins. The delivery of ubiquitin which conjugates to autophagosome is markedly enhanced, thereby the bacteria-containing vacuoles can be accumulated in autophagolysosomes and result in antimicrobial activity against MTB.

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ANTIBACTERIAL AUTOPHAGY REGULATION BY INNATE IMMUNE SIGNALING Immunity against intracellular mycobacteria primarily depends on the recognition of both the bacteria and their components by innate receptors on phagocytes and immune cells (39). However, pathogenic mycobacteria can successfully subvert or escape intracellular signaling pathways, thereby resisting the host innate immune machineries and hiding from autophagic degradation pathways (6). Innate receptors are known as pattern recognition receptors (PRRs), and include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptors (NLRs), depending on the cellular location or ligand recognition (40). PRR stimulation by mycobacterial products initiates and activates the innate immune response during mycobacterial infection. Although the evidence remains contradictory, TLR-mediated signaling has been thought to play an important role in host defense against MTB, especially in studies using mice that have been known to lack myeloid differentiation primary response gene 88 (MyD88) (41).

Innate receptors and autophagy Several TLRs and NLRs have been shown to be involved in the induction of autophagy, alone or in combination (3, 6). Although their ability to induce autophagy activation varies, stimulation of macrophages with soluble TLR agonists such as Pam3CSK4 (TLR1), Pam2CSK4 (TLR2), poly(I:C) (TLR3), lipopolysaccharides (LPS, TLR4) �agellin (TLR5), MALP-2 (TLR6), ssRNA (TLR7), and CpG oligodeoxy nucleotides (TLR9) can enhance the conventional autophagy in macrophages (42). Other reports show that Pam3CSK4 is fused with latex beads, but not with soluble ligand alone, and signi�cantly induces phagosomal maturation in primary macrophages (43). The combinatorial signals induced by zymosan, a TLR2/Dectin-1 agonist, have led to the rapid recruitment of LC3 to the phagosome in murine macrophages (43). Additionally, the intracellular sensor proteins Nod1 and Nod2 play a role in the induction of autophagosomes in response to bacterial infection, but not conventional rapamycin or nutrient starvation (44). These studies suggest that autophagy, innate signaling, and phagocytosis pathways interact to restrict microbial replication and mount the appropriate innate responses.

Innate signaling pathways and autophagy TLR4/LPS induction of autophagy is regulated through a TIR domain-containing adapter-inducing interferon-β (TRIF)-dependent pathway (45). Additionally, receptor-interacting protein (RIP1) and p38 mitogen-activated protein kinase (MAPK) are required for TLR4/LPS-induced autophagy (45). Furthermore, the LPS treatment augments the expression of the murine immunity-related GTPase

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LRG47 (46), which is crucial for the induction of autophagy and generation of autolysosomal organelles (8, 9). TLR stimulation also leads to autophagy induction through the interaction of Beclin-1 with the MyD88 and TRIF adaptor molecules, and a reduction in Beclin-1 binding to anti-apoptotic proteins Bcl-2 and Bcl-XL (47). However, the signaling pathways downstream of Nod1 and Nod2, which lead to the formation of autophagosomes, remain unclear (44).

During the response to pathogenic invasion, the autophagy pathway is required for controlling the aberrant activation of proin�ammatory responses and in�ammasome activation (48). In particular, the activation of nuclear factor (NF)-KB, a common downstream transcriptional factor of TLR signaling, has been shown to act as a potent negative regulator in the autophagy process (49, 50). Therefore, it is thought that a balance between the activating and suppressing signals during innate responses is necessary for autophagy induction, which also functions as a negative feedback signal that curbs excessive in�ammatory responses (50).

Notably, the induction of autophagy activation by different TLR causes enhanced co-localization of intracellular MTB with autophagosomes and the inhibition of intracellular mycobacteria (42, 45). For example, autophagic activation by ssRNA or imiquimod results in a 20-40% reduction in intracellular mycobacteria (42). These data suggest that the stimulation of autophagy by a variety of PRR signaling pathways, some of which are independent of mycobacteria or their components, is capable of eliminating intracellular microbes, including mycobacteria. However, the stimulation of primary cells with TLR ligands does not lead to autophagy activation (51). In addition, MHC class II-positive cell types such as dendritic cells, B cells, and epithelial cells have a constitutively high basal level of autophagy, thus enabling ef�cient delivery of cytosolic proteins for helper T cell stimulation (3, 52). Future studies will be needed to clarify the differential patterns and regulatory mechanisms of autophagy activation induced by a variety of mycobacterial antigens in various cell types, and ultimately in vivo.

Mycobacterial antigens and autophagy The induction of autophagy by mycobacterial antigens involves the regulation of innate immune signaling pathways. The mycobacterial TLR2/1 ligand, LpqH lipoprotein, robustly activates autophagosome and autolysosome formation in human monocytes/macrophages (13). LpqH-induced autophagy is dependent on intracellular calcium in�ux, AMP-activated protein kinase (AMPK), and p38 MAPK activation (13). More recent studies have shown that the ‘enhanced intracellular survival (eis)’ gene of MTB plays a role in the modulation of macrophage autophagy

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and in�ammation, since eis-de�cient MTB shows enhanced autophagy activation and proin�ammatory responses in vitro and in vivo (53). However, this upregulated autophagy activation does not lead to host defense, but has resulted in cell death caused by autophagy inhibition (53). This suggests that excessive autophagy and concurrent oxidative stress may not lead to host defense against mycobacterial infection (53). Further studies are needed to clarify the innate molecular mechanisms by which they ef�ciently cooperate with autophagy pathways to combat the intracellular mycobacteria.

IMMUNE EFFECTORS AND ANTIBACTERIAL AUTOPHAGY The mycobacterial cell wall and secreted antigens are key in host protective and in�ammatory responses through the induction of a variety of cytokines, the principal immune effectors during mycobacterial infection (54, 55). Mycobacterial glycolipids lipoarabinomannan (LAM), capped with phospho-inositol (PILAM), and lipomannans (LMs) are potent inducers of cytokine production by innate cells, such as tumor necrosis factor (TNF)-α, interleukin (IL)-8, and IL-12p40 (54). ManLAM, the other type of LAM, contributes to mycobacterial persistence through the inhibition of IL-12p40 production in macrophages and DCs (56). Numerous studies have reported that a variety of mycobacterial protein antigens are capable of inducing cytokines/chemokine production (55). Several cytokines, such as the IFN-γ/IL-12 axis or TNF-α, are known to be critical in host defense against mycobacterial infection, as previously demonstrated in human genetic and mouse studies (57, 58, 59). Although Th1 cytokines and TNF-α are essential for the control of MTB infection, their overproduction may result in unwanted immunopathologic responses (55, 59).

Importantly, Th1 cytokines (IFN-γ) and TNF-α activate autophagy, whereas Th2 cytokines (IL-4, IL-13) inhibit the autophagy pathway (8, 60, 61, 62). The activation of macrophages by IFN-γ results in enhanced mycobacterial phagosomal maturation and inhibits the survival of intracellular mycobacteria through autophagy (8). The IFN-γ-downstream effectors, such as immunity-related p47 guanosine triphosphatases (IRG), play an important role in host defense against intracellular pathogens, including mycobacteria (14, 63). The murine Irgm1 (LRG-47) GTPase and the human Irgm1 ortholog, IRGM, can induce autophagy and enhance the control of intracellular bacteria (8, 9, 14, 63). Moreover, murine Irgm1 has no signi�cant effect on the development of host responses in a Th2-polarized condition and

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promotes the survival of mature effector CD4+ T lymphocytes through protection from IFN-γ-induced cell death (64). Human genetic studies have shown that single nucleotide polymorphisms (SNP) in the Irgm gene are strongly associated with susceptibility to TB in different human populations (65, 66). Recently, another IFN-γ-inducible gene family, the 65-kilodalton (kD) guanylate-binding protein (Gbp) genes (Gbp1, Gbp6, Gbp7, and Gbp10), have been shown to play a critical role in host protection against listerial and mycobacterial infection through coordinating principal defense mechanisms, including the phagocyte oxidase, antimicrobial peptides, and autophagy effectors (10). These data suggest that Th1-type immune responses induced by IFN-γ promote host defense against mycobacteria through both autophagy and other effectors such as IRG and Gbp. However, IFN-γ itself increases both caspase-dependent DNA cleavage and caspase-independent damage of mitochondria, thus promoting apoptosis and necrosis (67, 68). In physiologic conditions with a high mycobacterial burden, IFN-γ may accelerate the spread of TB infection through the induction of caspase-independent cell death (67).

The induction and regulation of autophagy is a promising avenue for novel clinical applications such as the adjunctive treatment of TB and vaccine development (7, 69). In the vaccine area, autophagy-promoting strategies will be effective for the prevention of new TB infection or reactivation of latent TB (7, 69). Jagannath et al. have shown that autophagy affects the processing of the immunodominant mycobacterial antigen Ag85B in antigen-presenting cells infected with MTB, thereby enhancing the co-localization of mycobacterial phagosomes with autolysosomes (70). Mice immunized with rapamycin-treated dendritic cells (DCs) show enhanced Th1-induced protective immune responses during infection with virulent MTB (70). In the context of vaccine and/or therapeutic development using autophagy modulation, it should be noted that several immune molecules inhibit and suppress the autophagy pathway and antimicrobial responses. The Th2 cytokines IL-4 and IL-13 inhibit autophagy and autophagy-mediated killing of intracellular mycobacteria (60). In developing countries where the current Bacillus Calmette et Guérin (BCG) vaccine fails to protect against TB, the pre-existing IL-4 response blocks protective immunity and results in impaired antimicrobial responses (71). Indeed, this subversive effect of IL-4 (and IL-13) is associated with reduced TNF-α-induced apoptosis of infected cells, reduced iNOS activities, and increased expansion of antigen-speci�c FOXP-3+ regulatory T cells (71). Moreover, the Th2-like response can lead to immunopathologic effects during TB through eliciting TNF-α toxicity and the induction of pulmonary �brosis (72). Thus, the development

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of effective vaccines and therapeutics that affect autophagy activation requires a better understanding of the components, leading to a shift from the preexisting Th2 phenotype to a Th1 response. More comprehensive studies are essential to elucidate the roles and mechanisms of various immune effectors in autophagy activation during mycobacterial infection.

VITAMIN D, CALCIUM AND AUTOPHAGY IN MYCOBACTERIAL INFECTION Several anti-TB drugs are available, but TB remains a leading cause of morbidity and mortality where eight million new cases occur annually (73). Vitamin D was considered a good candidate for the treatment of TB- a result which was produced in a clinical trial run in 1946 (74). After treatment with oral vitamin D2, 18 of 32 patients with lupus vulgaris had been cured and 9 patients improved (74). Since then, numerous studies have suggested that vitamin D possesses several important anti-TB activities. Insuf�cient vitamin D serum levels were associated with the risk of TB (75, 76) and the administration of vitamin D to TB patients resulted in higher sputum conversion and radiological improvement compared with placebo (77).

Despite the fact that vitamin D treatment has bene�ts for TB, the mechanism of action has not been elucidated. It was suggested in 1986 (78) and 1987 (79) that vitamin D plays a critical role in antimicrobial activity in MTB-infected human monocytes and macrophages, since 1,25D treatment resulted in a decrease in intracellular bacterial survival in vitro. Further studies have reported that this 1,25D3-induced antimycobacterial activity was regulated by phosphatidylinositol 3-kinase and mediated by the nicotinamide adenine dinucleotide phosphate- oxidase (NADPH) dependent phagocyte oxidase in monocytes (80).

Previous studies have also reported that 1,25D functions against mycobacterial infection through the induction of the antimicrobial peptide cathelicidin in human macrophages and monocytes (11). In mammals, various antimicrobial peptides and precursors have been reported, including cathelicidin-derived AMPs, defensins, and histatins (81). Stimulation of macrophages with 1,25D induces the production of several antimicrobial peptides including cathelicidin (11) and DEFB4 (HBD2) (82), thereby increasing the killing effect. In addition to the direct stimulation by 1,25D, TLR2/1 activation induces the expression of VDR and Cyp27b1, which converts inactive provitamin D (25D) into 1,25D. TLR2/1–induced cathelicidin possesses

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antimicrobial activity against intracellular MTB (11). Similar to cathelicidin, HBD2 plays a role in antimycobacterial innate responses associated with vitamin D. HBD2 can be induced by MTB (82) or TLR2 (83) stimulation. Both HBD2 and cathelicidin are required for TLR2/1-induced antimicrobial activity against intracellular mycobacteria (84).

Induction of autophagy leads to co-localization between the increased mycobacterial phagosome and LC3 autophagosomes, and results in bacterial killing in macrophages (8). Moreover, the increase in intracellular calcium leads to a recovery in Ptdlns3P levels, a central target for autophagy, in phagosomes with live mycobacteria (85). Other studies have demonstrated that ATP-dependent autophagy is also calcium-dependent and is associated with an increase in the killing of intracellular BCG mycobacteria (86). 1,25D3, or its synthetic analogue EB1089, can induce autophagy in breast cancer cells. This effect is mediated by the Ca2+ release from the endoplasmic reticulum to the cytosol (87, 88). In addition, vitamin D3-induced autophagy and the co-localization of LC3 with human cationic antimicrobial peptide hCAP-18/LL-37 are Ca2+/calmodulin- kinase-dependent, kinaseb-dependent, and AMPK dependent processes in human macrophages and essential for the formation of autophagolysosomes and antimycobacterial activity (12, 87). These data collectively suggest that 1,25D activates cathelicidin generation and functions either directly as part of the antimicrobial pathway, or indirectly through the activation of autophagy. Furthermore, LpqH, a TLR2/1 agonist, activates autophagy and induces the production of cathelicidin through the activation of VDR signaling in human monocytes with suf�cient 25D (13). The Ca2+/CaMKK/AMPK/p38 MAPK signaling pathway is required for LpqH-induced Cyp27b1 expression (13).

HBD2 is activated in human monocytes by TLR2/1-induced IL-1β-dependent immune responses and is required for the antimicrobial activities of human primary monocytes (84). However, DEFB4 (HBD2) is not involved in the LpqH-induced autophagy activation (13). Therefore, these results clearly demonstrate that TLR2/1 signaling activates antibacterial defense molecules such as defensins and cathelicidin through IL-1β and functional vitamin D3 receptor-dependent autophagy pathways. These molecules are known to cooperate to mount an ef�cient innate immune response against mycobacteria.

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CARGO-RECEPTORS AND AUTOPHAGY Recently, several molecules such as p62/SQSTM1, NDP52, and NBR1 have been suggested to be autophagy receptors (also called cargo receptors). The discovery of autophagy receptors, which speci�cally recognize intracellular cargos fated for degradation, have supplied a new insight into autophagy. These receptors play a role in ubiquitin-proteasome degradation through tagging the target proteins with ubiquitin, and targeting autophagy molecules, including LC3/GABARAP and ATG5. These receptors are emerging as critical links between the autophagy and ubiquitination pathways, both of which are important for the degradation and removal of harmful proteins (aggregated- or misfolded-proteins) or organelles (dysfunctional- or damaged-organelles). There is increasing evidence of a link between the autophagy and proteasome pathways, as well as the roles of ubiquitin and ubiquitin-binding proteins in selective autophagy.

Autophagy activation in MTB-infected macrophages induces the delivery of ubiquitin and ubiquitin-derived peptides into bacterial phagosomes, followed by lysosome maturation (89). Recent studies demonstrated that two related mammalian proteins, p62/SQSTM1 and NBR1, act as cargo receptors for selective autophagy of ubiquitinated substrates (90, 91, 92). Autophagic degradation of p62/SQSTM is dependent on an 11-amino acid long, linear motif (called LC3-interacting region; LIR) that is involved in the interaction with the LC3/GABARAP family. Another cargo receptor, NBR1 (a neighbor of BRCA1 gene 1), has been much less studied as compared with p62/SQSTM1. NBR1 can also bind to Atg8 family members, targeting ubiquitinated substrates to autophagic machineries (91, 93, 94). NDP52 (nuclear dot protein 52), known as a myosin VI binding partner (95), binds ubiquitin, mediating an interaction with ATG8/LC3 and delivering cytosolic aggregates or bacteria into autophagosomes (96, 97). In addition to the LIR motifs found in p62/SQSTM1, NBR1 and NDP52 and other cargo receptors including NIX, calreticulin, and clathrin heavy chain, LIR motifs are used to bind to GABARAP (98, 99, 100). The relative contributions and in vivo relevance of these cargo receptors in the context of antibacterial autophagy during intracellular infection remain to be determined.

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CONCLUSION TB is an immune disease caused by mycobacteria, which can invade and reside within the host cell phagosomes. Virulent mycobacteria have developed various tactics to escape host defense mechanisms. Since evidence is accumulating that autophagy is a basic defense mechanism against pathogens, new approaches through autophagy activation are now being considered for improving protective immunity against mycobacterial infection. Thus, de�ning the molecular and cellular immune processes that result in the activation of autophagy is essential for these efforts. Several innate molecules, including PRRs along with their innate signaling partners/proteins, have been described as crucial regulators for mycobacterial eradication, through the induction or activation of the autophagy processes. Recent discoveries of the roles of a variety of immune effectors, including cytokines and IRG (p47 GTPases), have provided an insight into the connections between innate responses and autophagic mechanisms that result in ef�cient antimicrobial responses. Moreover, vitamin D-dependent innate immune activation is critically involved in host defense against mycobacterial infection. Vitamin D can induce host cell autophagy through cathelicidin-induced co-localization of autophagosomes and phagosomes, which leads to the killing of mycobacteria. Importantly, in 25D-suf�cient sera, TLR signaling is connected with functional vitamin D receptor signaling, leading to the activation of Cyp27b1, cathelicidin expression, and autophagy activation. In the cytosol, several adaptors such as p62, NBR1, and NDP52 play an important role in the autophagic uptake of intracellular pathogens, through recognition of targets via ubiquitination.

Overall, this review has summarized the current knowledge of antimycobacterial autophagy. In contemplating how these �ndings can lead to the development of novel therapies and/or vaccines, it is important to understand their effect on immune responses. Further knowledge of the roles of immune regulatory molecules that normally function to increase host defenses or modulate in�ammatory processes will provide information on the precise mechanisms of autophagy regulation during mycobacterial infection.

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